Coenzyme A (CoA) and its thioester derivative Acetyl CoA (A-CoA) are essential intermediates in numerous biosynthetic and energy yielding metabolic pathways as well as regulators of several key metabolic reactions. A-CoA is an important intracellular metabolite in central carbon metabolism and is a precursor in the enzymatic synthesis of many useful compounds. A-CoA is formed during the enzymatic oxidation of pyruvate or fatty acids, and from free acetate in the presence of the enzyme acetyl-CoA synthase. There are several key rate limiting steps in the biosynthesis of A-CoA. The overexpression of the enzymes catalyzing these rate limiting steps increases the intracellular levels of A-CoA. The A-CoA node serves as a connecting point at which several metabolic pathways intersect. Enhancing the A-CoA flux, i.e., the amount of A-CoA generated in a given time, through the A-CoA node is a useful strategy for increasing the production of compounds that require A-CoA for their biosynthesis.
CoA and A-CoA are precursors to many industrially useful compounds. A-CoA is also a substrates in alcohol acetyl transferase reactions that produce various acetate esters. In addition, A-CoA and its condensation product acetoacetyl-CoA are involved in the biological production of various polyhydroxybutyrates (PHBs). A-CoA can be carboxylated to malonyl-CoA and subsequently enter pathways to isoprenoid and terpenoid compounds through mevalonate. In sum, enhancing the intracellular pools/flux of A-CoA has implications in improving the production of the useful compounds derived from A-CoA.
Existing methodologies focus on the engineering of metabolic pathways by overexpressing enzymes that are directly involved in the production of a target compound. The invention claimed and described herein differs from existing methodologies in that in the present invention, cellular metabolism is altered to increase glycolytic flux and to direct this increased flux towards the production of precursor molecules such as A-CoA. The increased production of A-CoA in turn increases the production of target compounds such as esters, PHBs and polyketides.
Metabolic engineering has the potential to considerably improve process productivity by manipulating the throughput of metabolic pathways. Most current metabolic engineering studies focus on manipulating enzyme levels through the amplification, addition, or deletion of a particular pathway. However, cofactors play an essential role in a large number of biochemical reactions and their manipulation has the potential to be used, as an additional tool to achieve desired metabolic engineering goals. In addition, cofactor manipulation may also provide an additional means to study cellular metabolism, in particular the interplay between cofactor levels/fluxes and metabolic fluxes.